The nervous system contains two classes of cells: the nerve cells (or neurons) and neuroglia cells (or glia). These cells are distinguished by morphological, biochemical and functional differences. Morphologically, neurons have a cell body and projecting extensions or neurites (processes) of varying length. In vivo, neuritic extensions are further divided into axons (which transfer signals away from the neuron) and dendrites (which transfer signals to the neuron). Among many other biochemical and biophysical processes, neurons synthesize specific chemicals involved in signaling of information. In the central nervous system (CNS) glia are nine times more prevalent than nerve cells. Glia are thought to serve as neural supportive elements by providing nutrients, growth or survival factors and extracellular matrices. These cells are morphologically distinct from nerve cells and do not synthesize neurotransmitters.
To date, attempts to implant functional neuronal cells have largely been unsuccessful. Once the cells send out neurites in vivo, they are very difficult to transplant. The neurites become damaged during preparation of the implantation culture, leading to the death of the cells. In response to this problem, cells for implantation have been obtained from embryos at a point in the embryonic maturation process prior to neurite formation to eliminate cell death due to disruption of the neurites.
However, it is also undesirable to transplant fresh tissue. A period of time to evaluate the tissue prior to implantation, such as to determine whether the tissue is contaminated with a virus, is highly desirable. To hold the tissue for a period of time, the tissue must either be cultured in vitro or frozen. Fresh tissue is fragile, and does not respond well to freezing. The percentage of viable cells in the fresh tissue is greatly diminished by the freeze/thaw process so that a substantial percentage of the cells in the implant are nonviable. In response to the problems inherent in using fresh or fresh-frozen tissue, attempts to culture undifferentiated cells in vitro for implantation have been performed.
Since the initial discovery 80 years ago by Ross Harrison that nerve fibers can survive under tissue culture conditions, the literature has become inundated with reports using cultured nervous tissue. Neuronal cells in vitro also send out neurites, leading to the same problems of cell death upon disruption of the neurites as encountered with fresh tissue. Therefore, attempts to transplant cells prior to production of the neurites in vitro have been made.
The consensus in the reports regarding dissociated CNS tissue (single cell suspension) is that (1) tissue derived from CNS areas which are no longer displaying neuronal division in vivo will only support glial survival in vitro (see, for example, Hansson et al., Brain Research 300:9-18 [1984]) and (2) tissue derived from CNS areas still undergoing neuronal division in vivo will allow both neuronal and glial survival in vitro; those neurons will not proliferate, but can differentiate to varying degrees under in vitro conditions (see for example, Ahnert-Hilger et al., Neuroscience 17(1):157-165 [1986] and Boss et al., Dev. Brain Res., 36:199-218 [1987]).
Methods which produce cell cultures in vitro in which neuronal cells proliferate are being sought. The method should also minimize the cell loss due to disruption of neurites upon preparation of the cells for implantation. In particular, such cell cultures which would produce dopamine following implantation are highly desirable.